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the different tested species of A.flavus group with respect to tbe transfommtion reactions of progesterone. Comparative biotransformation results showed that ...
Folia Mlcrobio[ 45 (3). 243 247 (2000)

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Progesterone Side-Chain Degradation by Some Species of Aspergillusflavus Group M. EMAN MOSFAFA, A.A. ZOFIRI* BolanY Department. PaczdO' qf Sctence. ASSlUt UmversiO', .4ssmt. E~;ol

Received 1 September 1999 ABSTRACT, Seventy isolates belonging to 6 species and one variety of A.flavus group were shown to degrade the progesterone side-chain to yield A4-androstene-3,17-dione and testosterone The isolates of five species (A.flavo-furcatis, 4 /Tavtts. ,4 or)vae, A parasiticus and A tamarii) possessed enz),me systems catalyzing tbe opening of ring D and formed testololactone as final steroid metabolite in addition to their abilit~ to produce tire above mentioned two products I ll3-Hydroxy-k4-androstene-3,17-dionewas formed by only Aflavus and ,4. tamaril while l l~-hydroxytestosterone was produced by ,4.Jlavo-fitrcatis. A. parasiticus and A. subolivaceus. The chromatographic resolution of the mixture products obtained (when the selective isolate of each species reacted with I g of progesterone) revealed that 60-75 % of progesterone was converted into A4-androstene-3,17-dione(8-30 %),

testosterone (7-33 %), testololactone (14-37 %) and other products (3-40 %). The most bioconversion activity was exhibited by ,4. oo,zae, followed by ,4. parasiticus. The highest values of A4-androstene-3,17-dione (30 % of added progesterone) and testosterone (33 %) were formed by A.flavus var. columnaris while those of testololactone (37 %) were produced by ,4 oo'zae A systematic variation could be observed between the different tested species of A.flavus group with respect to tbe transfommtion reactions of progesterone. Comparative biotransformation results showed that essential differences exist between the tested species in this group: this biochemical differentiation may supplement the morphological and other physiological criteria used in the identification of the different species in the A. flavus group.

The importance of microbial biotechnology in the production of steroid drugs and hormones was realized for the first time in 1952 when Murray and Peterson o f Upjohn C o m p a n y patented the process o f 1 l c,-hydroxylation o f progesterone by a Rhizopus species (Murray and Peterson 1952). Since then, microbial transformations o f steroids have proliferated, and specific microbial transformation steps have been incorporated into numerous partial syntheses o f new steroids for evaluation as drugs and hormones. A variety of steroids are widely used as anti-inflammatory, diuretic, anabolic, contraceptive, antiandrogenic, progestational and anticancer agents as well as in other applications (Mahato and Garai 1997). Also, some steroids are used as anesthetics and antifertility agents (EI-Refai 1995). One o f the important microbial reactions on steroids is the microbial side-chain degradation o f 4-dehydro-3,20-diketosteroids o f pregnene series, leading to the production o f the androgen series which may be further used as intermediates for the preparation o f the Cl8 estrogens (EI-Refai 1995) while the chemical degradation o f the progesterone side-chain to the 17-keto structure via the intermediates testosterone acetate and testosterone represents the same reaction type without any practical importance (Kieslich 1985). A variety o f microbial strains have been employed for this purpose (Mahato and Banerjee 1985; Mahato et al. 1989; Mahato and Garai 1997). This investigation was designed to study the progesterone sidechain degradation properties of 70 isolates belonging to 6 species and one variety (10 isolates each) o f Aspergillusflavus group as well as the qualitative and quantitative analysis with chromatographic resolution of the major products obtained by the active isolate of each species or variety. The use o f these biotransformation properties as one of the diagnostic features in the identification of the tested A . f l a v u s group members was examined.

MATERIALS AND METHODS

Organisms. Seventy isolates belonging to 6 species and one variety (10 isolates each) o f Aspergillus /lavus group (isolated previously from different sources through routine work carried out in our mycological laboratory) were used. Cultivation. Fungal isolates were maintained on slopes o f C z a p e k - D o x agar (Smith and Onions 1983). Inocula were prepared from 7-d-old cultures as spore suspensions in 0.2 % (V/I~) aqueous Tween 80. Isolates were inoculated into 250-mL Erlenmeyer flasks each containing 50 mL liquid sterilized glucoseCzapek medium fortified with 1.0 % peptone and 0.2 % yeast extract (Zohri and Ismail 1994) and incubated

*The present address: Date Processing Plant~ Al-Ahsa Food Industries Co.. PO. Box 353 I, AI-Ahsa31982, Saudi Arabia.

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M EMANMOSTAFA

and A A Z O H R I

V o l 45

at 28 ~ on a rotary shaker (1.8 Hz, amplitude 70 mm: 2 d). Thereafter, 10 mg (in qualitative studies) or 1 g (in quantitative studies) of progesterone (dissolved in suitable volume of 96 % ethanol) was added to each flask and the fermentation was continued for another 3 d. Extraction. At the end of fermentation, the content of each flask (medium + mycelium) was homogenized in a high-speed blender (frequency 270 Hz) with two volumes of chloroform. The extraction was repeated three times to ensure that all transformation products were extracted. Combined chloroform extracts were washed with half their volume of a 5 % sodium hydrogen carbonate solution, followed by an equal volume of distilled water. The extract was dried on anhydrous sodium sulfate, filtered and distilled under vacuum to give a semi-solid residue. TransJbrmation products were identified by comparison with standards using thin-layer chromatoplates (Sallam et al. 1969) prepared on plastic sheets pre-coated with a thin film (0.25 ram) of silica gel. As solvent systems were used cyclohexane-chloroform-acetic acid ( 8 : 1 : 1 ) , toluene-petroleum-ether (fraction 40-60 ~ ( 2 : 2 : 1 ) , cyclohexane-acetone-chloroform ( 3 : 1 : 1 ) and dichloromethane acetone (4:1). The plates were then sprayed with 12/Kl (Tfimovfi et al. 1955) and chlorosulfonic acid-acetic acid (Waldi 1965) reagents. The semi-solid residue was dissolved in a minimum volume of benzene and then fractionated on a standard activated alumina column (Khallil and Eman Mostafa 1996). The following solvents (200 mL each) were used: n-hexane, n-hexane-benzene ( 1 : 1), benzene, benzene diethylether (4 : I ) and diethylether. The fractions containing similar products were collected and recrystallized from a suitable solvent. Compounds were identified by comparison ofmp, mixed rap, [aiD, IR- and UV-absorption spectra with standards and/or literature data. Different products were also analyzed by a Cecil CE 1200 HPLC with two CE I100 pumps (equipped with manual injector), at a flow rate of 2.5 mL/min. The column was packed with Lichrosorb RP-18 (5 nm). Cecil CE 1200 UV detector (A254) fitted with a standard flow cell was used; the monitor had a path length of 10 mm and a volume o f 8 mL. Elution was carried out with methanol-water (1 "4).

R E S U L T S AND D I S C U S S I O N

Seventy isolates belonging to 6 species and one variety (10 isolates each) ofA. flavus group were tested for the ability to degrade the side-chain of progesterone (Table I). All tested isolates proved to be active and degraded the progesterone side-chain to give more than one of the following compounds: A4-andro stene-3,17-dione, testosterone, testololactone, 1113-hydroxy-A4-androstene-3,17-dione and 11 f3-hydroxytestosterone. T a b l e I. Progesterone side-chain degradation by' different species ofA. f l a v u s group

Metabolitcs formed a Fungal species or variety

,q flavo-fitrcatis

A4-androstene 3,17-dione

testosterone

testololactone

11 ~3-hydroxyA4-androstene-3,17-dione -

+

+

+

BATISTA & MAIA ,4 l l a v u s I,INK b

+

+

+

, - 1 / l a v u s '~ar c o h m m a r i s

+

+

+ + + +

+ + + +

-

I 1[3-hydroxytestosterone

+ -

RAPEr. & FENNELL .4 o o ' z a e (AHLB) COHN ,4. p a r a s i t i c u s SPEARE ,4 s u b o l i v a c e u s THOM ,4. tamarii KITA

+ + +

+

a(+) Metabolites

detected: (-) metabolites were not detected. bAll tested isolates o f A f l m , u s h y d r o x y l a t e d progesterone at positions C-11 and C - I 7 and formed l l o . - h y d r o x y p r o g e s t e r o n e , 17(x-hydroxyprogesterone and 1 Ict, 1 7 c t - d i h y d r o x y p r o g e s t e r o n e (in addition to the formed m e t a b o l i t e s p r o d u c e d b}, s i d e - c h a i n cleavage)

k4-Androstene-3,17-dione and testosterone were formed by all isolates (compounds are formed by an oxidative splitting of the side-chain of progesterone in position C-17). A4-Androstene-3,17-dione is

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PROGESTERONE SIDE-CHAIN DFGRADATION BY .-t /Tav~.~ 245

usually {n some form of equ{(ibrium w{th testosterone. The same was described by EI-Refai (1995): A llavus "~ 9 degraded the progesterone side-chain to give ~ 4 -androstene-_,, 17-&one. The two compounds were previously recorded as products of progesterone transformation by various fungal species other than ,4. flavus group, such as Penicillium waksmani (E1-Refai 1995), Saprolegnia hypos,ha and S. parasilica (Khallil and Eman Mostafa 1996). Transformation of progesterone to testosterone by some isolates of Fusarium solani, IL o,gvsporum and F. moniliforme was also demonstrated (El-Refai 1995). Testololactone was produced by isolates of five species only (A.flavo-fi,rcalis, A. flavus, A. orvzae. A. porasiticus and A. tamarii; Table 1). After the oxidati'~e splitting of progesterone side-chain, opening of the O ring follows giving rise to testololactone as the final steroid metabolite (Baeyer-Villiger oxidation). Peterson et el. (1957) reported that testo/o[actone fs the most common fina~ product of the side-chain degradation of progesterone. Khallil and Eman Mostafa (1996) found that testololactone was the final product of side-chain degradation of progesterone by S hypog~,na and S parasitica Testololactone was formed from progesterone by the action o f A flavus (Peterson et al. 1953), ,4. ot3vae (Kondo 1960) and ,4. tamaril (Brannon el at. 1965). We described for the first time the production of testo[olactone from progesterone by A. flavo-furcatis and A. parasiticus. 1113-Hydroxy-A4-androstene-3,17-dione was formed in a small detectable amount only by ,4.flavus and ,4 tamarii, while l l~3-hydroxytestosterone was produced (also in a small amount) by .4.Jtavo-Jurcatis, ,4 parasiticus and A. subo/ivaceus. Some (ungal and bacterial species produced different hydroxylated derivatives of,~4-androstene-3,17-dione and testosterone as biotransformation products formed by cleavage of the acetyl side-chain of progesterone (Mahato et al~ 1989). All tested isolates of A. Jtavus were shown to hydroxylate progesterone at positions C-II and C-17 with the formation of 1 lc*-hydroxyprogesterone, 17ot-hydroxyprogesterone and 1 lc~,l 7c~-dihydroxyprogesterone in addition to their ability to exhibit oxidative splitting of the side-chain of progesterone. The hydroxylation of progesterone to different mono- and/or dihydroxyprogesterone derivatives was previously described in several Aspergillus species (Mahato et at. ]989; Eman Mostafa 1995; Mahato and Garai 1997). tn order to economize on the use of the steroid material, I g of progesterone was subjected to transformation by' one of the active isolates of each ofA.flavo-Jhrcatis, A. flavus, ,4. flavus var. columnaris, ,4. o~3'zoe, ,4, parasiticus, A. subolivaceus and A. tamarii. At the end of fermentation, extraction, washing, drying and concentration of the extracts were carried out. The semi-solid residue of each isolate was collected and fractionated using column chromatography The presence of unchanged progesterone ranged from 25 to 40 % of added substrate; the degree of conversion ranged between 60 and 75 %. The most active isolate was A. oryzae followed by A, parasiticus and A. flavus; a lower conversion was found in the isolate of A. ,tTm,o-Ji~rcatis (Table 11). "l'abl~e i I, Quantitative determination of progesterone transformation Metabo~iles formeci b

Unchanged Fungal species or variety a

progesterone b

% .4. flavo-furcatis ,4 flavus

40 31

&4-at~drostene-3.17-dione

testosleror~e

15 8

17 7

testolo~ac~,wne

c,'~hers c

23 14

5 40

.-1.fiavus vat. cohmmarts

37

30

33

-

-

.4 .4 A A

25 30 38 34

20 15 28 1,5

{8 22 26 ~6

37 29 32

4 8 3

orvzae parasmcus subolivaceus tamarzi

a&,e Table I bPer cent of added progesterone (1 g). CThe names of the other metabolites see Table I; their yields were calculated theoretically (total value of driver metabolites ~ 100 unchanged progesterone. ,~4-androstene-3.17-dione. testosterone, testololactone).

The n-hexane fractions contained only [ipoid impurities. Fractions eluted with n-hexane-benzene (1: 1) were evaporated and crystallized fiom methanol-chloroform (I : 1) to yield crystals (mp 128-129 ~ (a]t) + t95~ Tt~is compound was found to be unchanged progesterone (25-40 % of added progesterone for all tested fungal isolates). The solid residues eluted with benzene were repeatedly crystallized fi-om methanol-chloroform (1:1) to afford crystals (mp 137-139 ~ laid +115~ the identity of this substance

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with A4-androstene-3,17-dione was demonstrated (mp 138-140 ~ Mamoli and Vercellone 1937). Different isolates converted 8-30 % o f added progesterone into A4-androstene-3,17-dione. Higher transformation activity was found in A.flavus vat. columnaris; A. flavus transformed lower amounts of this substrate (Table If). The fractions eluted with benzene-diethylether ( 4 : 1 ) yielded after repeated crystallization from chloroform-methanol (1 : 1) crystals (mp 165-167 ~ [a]D +21~ the substance did not depress the mp o f testosterone standard (rap 167-169 ~ Mamoli and Vercellone 1937). The highest yield of testosterone (33 %) was achieved in A . f l a v u s var. columnaris, the lowest (7 %) in A.Jlavus (Table It). Combined fractions eluted with diethylether yielded upon crystallization from methanol-chloroform (1 : 1) crystals (rap 206 207 c, [c~]D +460): the product was found to be testololactone, since no depression in the mp was observed upon mixing with an authentic sample oftestololactone (mp 210-212 ~ [a] D +43~ Peterson et al. 1953). Testololactone was produced by five species with a yield of 14-37 %. These yields were higher than those found with A4-androstene-3,17-dione and testosterone by the same isolates. A. oo,zae formed the highest amount while A. flavus produced the lowest one (Table I1). Progesterone, A4-androstene-3,17-dione, testosterone and testololactone gave IR spectra and HPLC retention times identical with authentic samples. These products were formed as the major part of detectable metabolites which were formed as a result of progesterone bioconversion. Other metabolites (which were produced in very low amounts and by a few members o f the tested group) were detected by TLC and their yields were only calculated (Tables I and ll) without determination o f rap, mixed rap, [a]D, IR- and UVabsorption spectra. We demonstrated that the transformation o f progesterone may be useful as one of the diagnostic biochemical features in the taxonomy o f several fungal genera, e.g., Fusarium ((~apek and Hane 1960), Penicillium (Capek and Hane 1962), Alternaria, Cladosporium and Stemphylium ((~apek et al. 1974), A niger group (Eman Mostafa 1995) and several others (cf. Zohri and Abdel-Galil 1999). We observed a systematic variation between the species o f A . f l a v u s group with respect to the transformation products of progesterone (see Table I). All the species or varieties had the capability o f degradation o f progesterone side-chain with the formation of A4-androstene-3,17-dione and testosterone. A. flavus vat. columnaris transformed progesterone into A4-androstene-3,17-dione and testosterone; A. o~yzae formed testololactone and A. subolivaceus produced 1 I f3-hydroxytestosterone besides the above mentioned two products. Each o f A. parasiticus and A. flavo-/itrcatis permit an oxidative splitting of the side-chain o f progesterone with the formation o f testololactone and l l{3-hydroxytestosterone besides A4-androstene-3,17-dione and testosterone. A.Jlavus and A. tamarii transformed progesterone into A4-androstene-3,17-dione, testosterone, testololactone and l l~3-hydroxy-A4-antrostene-3,17-dione. On the other hand, only A. jlavus possesses the enzyme systems catalyzing the formation o f 11 of and 17c~-monohydroxyprogesterone and 1 I c~, 17cx-dihydroxyprogesterone. Comparative biotransformation reactions of progesterone showed that essential differences exist between the different species of A. flavus group, where the different isolates o f individual species produce one or more identical products. This biochemical differentiation may supplement the morphological and physiological criteria used in the identification of the different species o f the ,4.flaw,s group. REFERENCES

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